Synthesis and spectroscopic characterization of photo-affinity peptide ligands to study rhodopsin-G protein interaction

Article abstract of DOI:10.1111/j.1751-1097.2008.00304.x

G protein-coupled receptors (GPCRs) are involved in the control of virtually all aspects of our behavior and physiology. Activated receptors catalyze nucleotide exchange in heterotrimeric G proteins (composed of α-GDP, β and γ subunits) on the inner surfa

Full text of DOI:10.1111/j.1751-1097.2008.00304.x

Photochemistry and Photobiology, 2008, 84: 831–838
Synthesis and Spectroscopic Characterization of Photo-affinity Peptide
Ligands to Study Rhodopsin–G Protein Interaction†
Yihui Chen‡1, Rolf Herrmann‡§2, Nathan Fishkin–1, Peter Henklein3, Koji Nakanishi*1 and Oliver P. Ernst*2
1Department of Chemistry, Columbia University, New York, NY
2
´
¨
Institut fu¨r Medizinische Physik und Biophysik, Charite-Universitatsmedizin Berlin, Berlin, Germany
3
´
Institut fu¨r Biochemie, Charite-Universitatsmedizin Berlin, Berlin, Germany
¨
Received 31 October 2007, accepted 11 December 2007, DOI: 10.1111 ⁄ j.1751-1097.2008.00304.x
processes. Accordingly, numerous drugs from the past and
present target membrane-bound GPCRs (3).
ABSTRACT
G protein-coupled receptors (GPCRs) are involved in the control
of virtually all aspects of our behavior and physiology. Activated
receptors catalyze nucleotide exchange in heterotrimeric G
proteins (composed of aÆGDP, b and c subunits) on the inner
surface of the cell membrane. The GPCR rhodopsin and the G
protein transducin (Gt) are key proteins in the early steps of
the visual cascade. The main receptor interaction sites on Gt are
the C-terminal tail of the Gta-subunit and the farnesylated
C-terminal tail of the Gtc-subunit. Synthetic peptides derived
from these C-termini specifically bind and stabilize the active
rhodopsin conformation (R*). Here we report the synthesis of
R*-interacting peptides containing photo-reactive groups with a
specific isotope pattern, which can facilitate detection of cross-
linked products by mass spectrometry. In a preliminary set of
experiments, we characterized such peptides derived from the
farnesylated Gtc C-terminus (Gtc(60-71)far) in terms of their
capability to bind R*. Here, we describe novel peptides with
photo-affinity labels that bind R* with affinities similar to that of
the native Gtc(60-71)far peptide. Such peptides will enable an
improved experimental strategy to probe rhodopsin–Gt interac-
tion and to map so far unknown interaction sites between both
proteins.
In visual signal transduction in rod photoreceptors, the
GPCR rhodopsin with its covalently bound chromophore
11-cis-retinal acts as the photon detector. Light absorption
causes cis fi trans isomerization and thereupon in situ forma-
tion of an activating ligand in rhodopsin’s chromophore binding
site. Rhodopsin and its cognate G protein transducin (Gt)
represent the only GPCR ⁄ G protein system for which crystal
structures are available (4–7). Different models of receptor ⁄ G
protein interaction were proposed on the basis of a variety of
experimental and theoretical approaches (8–22). However, the
detailed molecular mechanism of signal transfer between these
two proteins leading to nucleotide exchange is not clear. Several
putative interaction sites on Gt have been reported but only two
of them (depicted in orange in Fig. 1), i.e. the C-terminal tail of
Gta (Gta(340–350)) and the farnesylated C-terminal tail of Gtc
(Gtc(60–71)far), have been shown to specifically bind and
stabilize the active conformation of rhodopsin (R*), called
metarhodopsin II (MII) (18,23). Synthetic peptides correspond-
ing to Gta(340–350) (IKENLKDCGLF) and Gtc(60–71)far
(DKNPFKELKGGC-farnesyl) mimic the Gt holoprotein and
bind in a similar manner to MII. NMR studies showed that both
peptides adopt helical conformations upon receptor binding
(24,25). In Fig. 1, additional potential binding sites on Gt are
facing toward the cytoplasmic interaction surface of rhodopsin
(for a review, see Hamm [11]). Gt’s C-terminal a5 helix with the
Gta(340–350) tail was identified as the key determinant for Gt-
binding to R* and GDP release from Gta (12,18,21,26,27). It was
proposed that Gtc(60–71)far constitutes together with the
myristoylated Gta N-terminus (myristate shown in gray in
Fig. 1) an amphiphilic microdomain. This microdomain forms a
docking complex with R* upon the initial encounter (20). Upon
docking, Gta(340–350) becomes available for key interaction
with R*, resulting in the termination of the R* ⁄ Gtc(60–71)
interaction (19,20).
INTRODUCTION
G protein-coupled receptors (GPCRs) are one of the most
versatile families of proteins and respond as cell-surface
receptors to endogenous signals in the form of chemically
diverse small molecules. Ligand binding to the receptor
stabilizes the active receptor conformation (R*), which cata-
lyzes GDP fi GTP exchange in heterotrimeric G proteins
(composed of aÆGDP, b and c subunits) on the inner surface of
the cell membrane (1,2). The GPCR superfamily plays an
important role in many physiologic and pathophysiologic
Although modeling approaches led to various 3D models of
the R*ÆGt complex with different R* ⁄ Gt stoichiometries
including rhodopsin mono-, di- or tetramers (28–31), the
relative orientation of Gt and R* is not known. However,
rhodopsin monomers were identified as the smallest functional
receptor unit capable of catalyzing nucleotide exchange in Gt
(32,33). Khorana and coworkers used a crosslinking approach
to probe the relative orientation of Gt and R*. They modified
†This invited paper is part of the Symposium-in-Print: Photoreceptors and
Signal Transduction.
‡Yihui Chen and Rolf Herrmann contributed equally to this study
§Current address: Rolf Herrmann, Duke University Eye Center, Durham, NC.
–Current address: Nathan Fishkin, Immunogen, Inc., Cambridge, MA.
*Corresponding authors email: kn5@columbia.edu (Koji Nakanishi),
oliver.ernst@charite.de (Oliver P. Ernst)
ꢀ 2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/08
831

832 Yihui Chen et al.
MATERIALS AND METHODS
General experimental procedure. THF and diethyl ether were distilled
from sodium ⁄ benzophenone ketyl. Methylene chloride was distilled
over CaH2. All other reagents and starting materials were purchased
and used as received (Aldrich, St. Louis, MO). Reactions were
monitored by analytical TLC using silica gel 60 F254 plates and spots
were visualized by UV (254 nm) or oxidative stain (PMA). Flash
column chromatography was performed using silica gel (230–400
mesh). Analytical HPLC was performed on a Waters instrument with
UV detector (200–700 nm) using
a C18 reverse-phase column
(4.6 mm · 150 mm; Waters) to analyze synthetic Gtc-derived peptides.
Water and acetonitrile were used as eluents in various gradient
1
programs at a flow of 0.6 mL min)1. H-NMR spectra were recorded
with Bruker (300 or 400 MHz) spectrometers. The chemical shifts are
expressed in p.p.m. (d) downfield from tetramethylsilane (in CDCl3).
UV–Vis spectra were recorded at 25ꢁC in a Perkin-Elmer Lambda 40
spectrophotometer. Mass spectra were measured on a JEOL KMS-
HX110 ⁄ 100A HF mass spectrometer under FAB conditions.
Peptide synthesis. Peptide synthesis was carried out on a TentaGel
resin (Rapp Polymere, Tubingen), building up the sequence from the
C-terminus to the N-terminus using Fmoc-protected amino acids.
After purification, peptides were lyophilized and stored at )20ꢁC under
nitrogen. Immediately before the experiments, the peptides were
dissolved in appropriate buffers or deionized water and adjusted to pH
7 to obtain stock solutions of 1–10 mM. Peptide farnesylation was
carried out as follows: 5 lM peptide was dissolved in 50% n-propyl
alcohol and Na2CO3 was added to a final concentration of 20 mM. The
solution was then treated with 30 lM farnesyl bromide in a 10%
solution of n-propyl alcohol for 18–24 h at room temperature in the
dark. Farnesylated peptides were purified by reversed phase chroma-
tography using a PepRPC fast protein liquid chromatography column
(Pharmacia Biotech, Inc.) using
a linear (0–100%) gradient of
acetonitrile in water containing 0.1% trifluoroacetic acid (TFA). The
molecular weight of purified peptides was determined by electrospray
mass spectrometry using a Vestec VT 201 mass spectrometer.
Meta II stabilization assay. The amount of ‘‘Extra MII’’ was
monitored by time-resolved UV–Vis spectroscopy using
wavelength spectrophotometer (Shimadzu UV300). Recorded traces
represent readings of an absorbance difference (A380nm ) A417nm
a dual
Figure 1. Interface between the heterotrimeric G protein transducin
(Gt) and its receptor rhodopsin. Gt (pdb entry 1GOT) consists of the
nucleotide-binding Gta subunit and the Gtbc heterodimer. The GPCR
rhodopsin (pdb entry 1L9H) contains the chromophore 11-cis-retinal
(gray, space-filling model) linked via a Schiff base to Lys-296. The lipid
modifications and the C-terminal regions of Gta and Gtc as well as the
N-terminus of Gta were modeled (19). C-terminal regions shown in
orange are key interaction sites for R*.
;
absorption maximum of MII and isosbestic point between MI and
MII) from the samples containing 10 lM of washed rod outer segment
membranes (prepared as in Herrmann et al. [19]). All measurements
were made in 100 mM Na-Hepes pH 7.9, 50 mM NaCl, 1 mM DTT,
1 mM MgCl2, 1 mM EDTA at 4ꢁC. Cuvette path length was 2 mm.
Twelve percent of rhodopsin was flash-activated by 500
20 nm
light. Values of half-maximal saturation (EC50 values) were obtained
by fitting the dose–response curves to the following equation:
y = Axn ⁄ (EC50n+xn), with x as the peptide concentration, y the
amount of ‘‘Extra MII,’’ i.e. the amplitude difference between the
peptide or Gt induced ‘‘Extra MII’’ signal and the reference signal (no
peptide or Gt added). A is the amount of maximal ‘‘Extra MII’’ formed
and n the Hill coefficient. Except for the data resulting from peptide 4
(Fig. 5, n = 1.5), n was set to 1 in all fits.
Benzaldehyde (d5) 6. To a stirred solution of 468 mg (6 mmol)
benzene (d6) in 10 mL of dichloromethane, 1.52 g (12 mmol) of
titanium (IV) chloride was added at 0ꢁC in very slow speed, then
followed by drop-wise addition of 690 mg (6 mmol) freshly distilled
dichloromethyl methyl ether (Scheme 1). After 25 min, the reaction
mixture was warmed up to room temperature, stirred for another 1 h
and poured into 10 g ice water. The mixture was extracted with
dichloromethane three times, and the combined organic layers were
washed with 5% aqueous NaHCO3 and dried over sodium sulfate. The
residue was purified by chromatography on silica gel (hexane:ethyl
acetate 6:1) to give the pure compound 550 mg (81%). 1H NMR
(CDCl3, 300 MHz): d 10.00 (s, 1 H); MS m ⁄ z 107.4, [M +H+] calcd.
for C7D5H1O 106.1.
particular cysteines on rhodopsin’s cytoplasmic surface with
two different heterobifunctional crosslinking reagents, a pho-
toactivatable and a chemically preactivated one (34,35). In
their study, Gt could be crosslinked from a variety of different
sites on rhodopsin’s second and third cytoplasmic loops with
each of these reagents. This was consistent with earlier reports
that these loops are involved in G protein interaction (for a
review, see Hamm [11]). When Cys-240 on rhodopsin’s third
cytoplasmic loop was modified with a crosslinker, the cross-
linked counterparts on Gt were analyzed using mass spec-
trometry and identified as amino- and carboxyl-terminal
regions of Gta. However, no crosslinking products could be
identified for the C-terminal region of Gtc, leaving the question
open as to where this Gt key binding site interacts with R*.
Therefore, our study aims to apply the photoaffinity labeling
approach to the Gtc(60–71)far binding site. Here we report the
design of novel, suitably modified peptides corresponding to
Gtc(60–71)far containing isotope-coded photolabile groups to
facilitate receptor fragment identification by mass spectrom-
etry. We discuss the synthesis and present R* binding data of
these peptides.
(2S,5R)-2-(4-bromobenzyl)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-
pyrazine 7. To
a solution of (R)-2-isopropyl-3,6-dimethoxy-2,5-
dihydropyrazine (1.5 g, 8 mmol) in THF (25 mL), was added
n-BuLi (4.2 mL, 1.5 M in hexane) at )78ꢁC drop-wise. After stirring
for 15 min, a solution of 4-bromobenzyl bromide (2.5 g, 10.0 mmol)
in THF (4 mL) was added drop-wise. The resulting mixture was
stirred for 3 h at )78ꢁC. After removal of THF, the residue was

Photochemistry and Photobiology, 2008, 84 833
Scheme 1. Enantioselective synthesis of Fmoc-protected 4-benzoyl-L-phenylalanine.
diluted with methylene chloride (60 mL). The organic layer was
washed with 5% sulfuric acid, water and brine, then dried and
concentrated in vacuo. Purification by chomatography on silica gel
(ethyl acetate:hexane = 1:7 by volume), afforded compound 7 as a
(S)-methyl 2-amino-3-(4-(phenylcarbonyl)phenyl)propanoate (d5)
10. TFA (3.3 mmol, 0.1 N in water, 3 equiv.) was added to a solution
of compound 9 (0.40 g, 1.1 mmol) in a mixture of methylene chloride
(3 mL) and acetonitrile (30 mL). The resulting mixture was stirred at
RT for 3 h. Acetonitrile and TFA were removed in vacuo to produce a
suspension of the methyl ester 10 and (L)-valine methyl ester as TFA
salts in water. This suspension, without neutralization with base, was
then extracted five times with methylene chloride. The combined
organic phase was dried and concentrated in vacuo to give 0.29 g
(99%) of the amino ester 10 as a white powder. This procedure
effectively eliminated the TFA salt of (D)-valine methyl ester which
remained in the aqueous phase: 1H NMR (CDCl3, 300 MHz) d8.45
single diastereomer (100% de, 2.54 g, 90%) as
a
white solid:
7.31
25
½aꢀD = 40.2 (CH2Cl2); 1H NMR (CDCl3, 300 MHz)
d
(d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 4.28 (dd, J = 8.7,
3.5 Hz, 1H), 3.70 (s, 3H), 3.66 (s, 3H), 3.40 (t, J = 3.5 Hz 1H),
3.03 (d, J = 4.8 Hz, 2H), 2.15 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H),
0.61 (d, J = 6.8 Hz, 3H); MS m ⁄ z 353.1, [M 100%] calcd. for
C16H21N2O2 353.3.
(4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)
methyl)phenyl)(phenyl)methanol (d5) 8. t-Butyllithium (4.65 mL,
7.8 mmol, 1.7 M in pentane) was added drop-wise at )78ꢁC to a
solution of bromide 7 (1.65 g, 4.65 mmol) in distilled THF (50 mL).
After 15 min, a solution of aldehyde 6 (1.0 g, 9.3 mmol) in distilled
THF (8 mL) was slowly cannulated into the reaction mixture. The
mixture was stirred for an additional 4 h at the same temperature. A
saturated ammonium chloride solution was added and the mixture
warmed to RT. The volatiles were removed in vacuo and the residue
taken up in ethyl acetate and washed once with water. The aqueous
layer was extracted three times with ethyl acetate. The combined
organic extracts were dried, concentrated, and the residue purified by
chromatography on silica gel (hexane:ethyl acetate 8:1) to furnish the
_
(br, 2H, NH2) 7.68 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H),
4.30 (t, J = 6.6 Hz, 1H), 3.68 (s, 3H), 3.35 (d, J = 6.6 Hz, 2H); MS
m ⁄ z 289.4, [M 100%] calcd. for C17D5H18NO3 289.3.
(S)-2-amino-3-(4-(phenylcarbonyl)phenyl)propanoic acid (d5) 11.
(S)-methyl
2-amino-3-(4-(phenylcarbonyl)phenyl)propanoate
10
(0.29 g, 1.0 mmol) was dissolved in 50 mL of THF. An aqueous
solution of LiOHÆH2O (115 mg, 3.0 mmol) in 10 mL of water was
added at RT and the mixture stirred for 4–5 min. Following
neutralization of the mixture with 6 N HCl to pH = 3, THF was
evaporated in vacuo. A white precipitate of the amino acid-HCl salt
appeared on concentration that was cooled to 0ꢁC for 5 h, then
filtered, washed with chilled water two times and ether three times, and
dried under high vacuum over P2O5 overnight (yield = 0.29 g, 99%):
1H NMR (CD3OD, 300 MHz) d 7.93 (d, J = 8.1 Hz, 2H), 7.65
(d, J = 8.1 Hz, 2H), 3.58 (dd, J = 10.0, 4.6 Hz, 1H), 3.34
(d, J = 8.4 Hz, 1H), 3.30 (d, J = 8.4 Hz, 1H); MS m ⁄ z 275.0,
[M +H+] calcd. for C16D5H15NO3 274.3.
1
desired diastereomeric alcohols 8 (975 mg, 55%) as a colorless oil: H
NMR (CDCl3, 300 MHz) d 7.20 (d, J = 8.1 Hz, 1H), 7.04
(d, J = 8.1 Hz, 1H), 6.90 (d, J = 8.5 Hz, 1H), 6.62 (d, J = 8.5 Hz,
€
1H), 5.78 (d, J = 3.1 Hz, 1H), 4.28 (m, 1H), 3.70 (d, J = 4.9 Hz, 3H),
3.66 (d, J = 3.2 Hz, 3H), 3.27 (dd, J = 10.8, 3.4 Hz, 1H), 3.06
(d, J = 4.8 Hz, 2H), 2.40 (d, J = 3.6 Hz, OH), 2.14 (m, 1H), 0.94
(m, 3H), 0.61 (dd, J = 4.5, 2.2 Hz, 3H); MS m ⁄ z 386.1, [M +H+
calcd. for C23D5H28N2O3 385.2.
]
RESULTS AND DISCUSSION
(4-(((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)
methyl)phenyl)(phenyl)methanone (d5) 9. Manganese dioxide (3.0 g,
34.5 mmol) was added in one portion at 0ꢁC to a solution of
benzylated alcohol 8 (0.45 g, 1.2 mmol) in methylene chloride (50 mL).
The mixture was stirred overnight at RT, then filtered through Celite
and concentrated in vacuo to yield 421.5 mg (99%) of ketone 9 as white
crystals: 1H NMR (CDCl3, 300 MHz) d 7.68 (d, J = 8.3 Hz, 2H), 7.23
(d, J = 8.3 Hz, 2H), 4.36 (dd, J = 5.1, 2.5 Hz 1H), 3.73 (s,, 3H), 3.68
(s, 3H), 3.44 (t, 3.4 Hz, 1H), 3.20 (d, J = 5.3 Hz, 2H), 2.18 (m, 1H),
0.97 (d, J = 6.8 3H), 0.64 (d, J = 6.7 Hz, 3H); MS m ⁄ z 390.5,
[M +H+] calcd. for C23D5H26N2O3 388.7.
Photolabile groups at the N-terminus of the Gtc(60-71)far
peptide
The native Gtc(60-71)far peptide is shown in Fig. 2. Photola-
bile analogs were prepared with a photolabile benzophenone
group either at position A, the peptide’s N-terminus, or at
position B, Phe-64. Position A was chosen because the
aspartate amino group of Gtc(60-71)far could be directly

834 Yihui Chen et al.
Figure 2. The Gtc(60-71)far peptide (DKNPFKELKGGC-farnesyl) is modified to contain a photolabile benzophenone at positions A or B.
coupled to commercially available p-benzoyl-L-phenylalanine
(Bpa) or 4-(para-bromobenzoyl)benzoic acid during peptide
synthesis. After deprotection, resin removal and farnesylation,
the respective photolabile peptides 1 and 2 were in hand
(Fig. 3). The 4-(para-bromobenzoyl)benzoic acid group in
peptide 2 was chosen to introduce a marker for mass spectro-
metric analysis. Crosslinking products containing this peptide
would yield double peaks split by 2 m ⁄ z units due to the
bromine isotope pattern.
To obtain peptide 3, 4-benzoylbenzoic acid was coupled to
(S)-malic acid-modified Gtc(61-71) on the resin (malic acid
replacing Asp-60), followed by deprotection, resin removal
and farnesylation. An ester linkage of the photolabile group
could allow aminolysis and e.g. amidation with 5-amino-
methylfluorescein for fluorescent labeling of crosslinking
products.
We tested the binding affinities of peptides 1–3 to light-
activated rhodopsin by using the ‘‘Extra MII’’ stabilization
Figure 3. Derivatives of the Gtc(60-71)far peptide with benzophenone modifications at the N-terminus.

Photochemistry and Photobiology, 2008, 84 835
sufficiently high concentrations of peptide (250 and
assay (36,37). Flash activation of rhodopsin leads to an
equilibrium between the inactive photoproduct metarhodopsin
I (MI) and its successor, the active form metarhodopsin II
(MII) that is capable of binding and activating Gt. Under the
conditions of the ‘‘Extra MII’’ assay (4ꢁC, pH 8), the MI ⁄ MII
equilibrium is on the side of MI. Binding of added Gt or
peptides derived from the C-termini of Gta or farnesylated Gtc,
respectively, stabilize MII. They do so at the expense of MI
and thereby shift the MI ⁄ MII equilibrium to the side of MII
(Extra MII) (36,37). As MI and MII can be distinguished
based on their different absorption maxima (MI at 480 nm and
MII at 380 nm), stabilization of MII is monitored by detecting
the absorption difference DA380)DA417 (‘‘Extra MII’’; 417 nm
representing the isosbestic point between MI and MII). Using
this spectral assay, we investigated the MII affinities of the
synthesized peptides 1–3 (Fig. 4). Unmodified Gtc(60-71)far
peptide stabilized MII in a concentration-dependent manner
(Fig. 4a) with an EC50 value of 91 lM, in agreement with
earlier determinations (17,18). Peptides 1 and 2 failed to
stabilize MII at similar concentrations (Fig. 4c,d), indicating
that N-terminal modification of Gtc(60-71)far may lead to a
loss of interaction with MII. Although unspecific binding to
the G protein cannot be excluded, it is conceivable that for
these peptides the photoproduct specificity is changed, i.e. that
they also recognize MI or earlier photoproducts even including
the ground state of rhodopsin. Competition experiments in
which peptide 2 competed against Gt in the ‘‘Extra MII’’ assay
2
500 lM) MII stabilization by Gt was significantly reduced.
Interestingly, the rate of MII formation (see the rising phase of
the traces in Fig. 4c) is decreased when peptide 2 was added
(magenta and yellow traces compared to green trace in
Fig. 4c). This observation further supports the conclusion
that peptide 2 binds to rhodopsin photoproducts other than
MII or the rhodopsin ground state. By doing so, the fraction of
MI available to react into the MII species is reduced, and
therefore the apparent MII formation rate is decreased.
In the case of peptide 3, ‘‘Extra MII’’ was formed with an
EC50 value of about 20 lM. However, the maximal amplitude
of the ‘‘Extra MII’’ signal under saturating peptide concen-
trations was only about 20% compared to the Gtc(60-71)far
(Fig. 4b,d), suggesting that the preference for MII is reduced,
however, in a far less pronounced manner compared to
peptides 1 and 2. Interestingly, also peptide 3 lacking the
benzophenone ester showed a reduced ‘‘Extra MII’’ amplitude
(data not shown), indicating that the replacement of Asp-60 by
malic acid affects the MI ⁄ MII specificity of Gtc(60-71)far as
well.
Substitution of Phe-64 in Gtc(60-71)far with
benzoylphenylalanine
In order to circumvent the problems with poor MII stabiliza-
tion of the N-terminally modified peptides, Phe-64 in Gtc(60-
71)far was considered as a position for introduction of a
indicate such
a behavior (Fig. 4c). In the presence of
Figure 4. Probing the rhodopsin binding properties of modified Gtc(60-71)far peptides containing a benzophenone moiety at the N-terminus by the
‘‘Extra MII’’ assay. Stabilization of MII by increasing concentrations of the native Gtc(60-71)far peptide (a) or peptide 3 (b) is measured as an
increase in relative UV–Vis absorption (absorption change at 380 nm minus the absorption change at 417 nm) after flash activation of rhodopsin
(flash symbol). MII binding of Gt (upper trace, green) is inhibited in the presence of peptide 2 (magenta and yellow) which fails to stabilize MII
by itself (c, bottom trace, red). Dose–response curves of the ‘‘Extra MII’’ data for all peptides tested (d). Plotted are ‘‘Extra MII’’ amplitude
versus peptide concentration. No MII binding is observed for peptides 1 and 2. Data are normalized to the maximal amplitude obtained for the
Gtc(60-71)far peptide.

836 Yihui Chen et al.
Figure 5. Gtc(60-71)far peptides modified at position Phe-64, which is replaced by 4-benzoyl-L-phenylalanine (Bpa).
para-benzoyl group by substitution with Bpa (4-benzoyl-L-
phenylalanine). Such a replacement is appealing as it would
represent a relatively small modification of the native peptide.
In order to facilitate mass spectrometric analysis of future
photocrosslinking products, attention was turned to the
synthesis of peptides 4 and 5, a pentadeuterated version of
peptide 4 (Fig. 5). Mixing both peptides in a 1:1 ratio for
photocrosslinking experiments with R* should facilitate iden-
tification of crosslinked products by the occurrence of double
peaks split by 5 m ⁄ z units due to the isotope coding. Peptide 4
was synthesized using commercially available Fmoc-4-benzoyl-
L-phenylalanine. For synthesis of peptide 5, a corresponding
pentadeuterated Fmoc-4-benzoyl-L-phenylalanine building
block was made (Scheme 1; for synthesis details, see Materials
and Methods).
To synthesize pentadeuterated 4-benzoyl-L-phenylalanine,
we used a bulky chiral ester to control diastereoselectivity (38).
The entire synthesis of 4-benzoyl-L-phenylalanine was carried
out in a straightforward manner using the commercially
available chiral auxiliary known as Schollkopf’s reagent
(Scheme 1). Thus, for the synthesis of peptide 5, p-bromo-
benzyl bromide was coupled to (R)-Schollkopf’s reagent, (2R)-
2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine, to give (S,R)
bislactim ether 7, which was obtained as a single diastereomer
(100% de) as indicated by the NMR and TLC analyses of the
crude isolate after work-up. Halogen-metal exchange of 7 at
)78ꢁC followed by the addition of pentadeuterated benzalde-
hyde 6 (PDB, obtained in 81% yield) gave a mixture of
diastereomeric benzhydryl alcohols (compound 8), which, after
purification, could be readily oxidized to benzophenone 9.
Mild acid hydrolysis of 9 to aminoester 10 was followed by a
very rapid reaction with a 1 M excess of LiOH in H2O ⁄ THF to
give amino acid 11; the Fmoc-protected amino acid 12 was
obtained by reaction of Fmoc-OSU with compound 11 and
used for synthesis of peptide 5.
Phe-64 was shown to be important for interaction of
Gtc(60-71)far with R* (17,39–41). Therefore, we tested the
binding affinities of peptide 4 to light-activated rhodopsin by
using the ‘‘Extra MII’’ assay as described above. Peptide 4
stabilized MII in a concentration-dependent manner with an
EC50 value of 60 lM (Fig. 6). This value was approximately
30% lower compared to the parent Gtc(60-71)far peptide
(EC50 = 91 lM), indicating a slight affinity increase in pep-
tide 4 for MII. Importantly, the para-benzoyl modification of
the Gtc(60-71)far peptide at position 64 showed little effect on
the amplitude of the ‘‘Extra MII’’ signals, indicating that the
specificity of the peptide for MII was not affected. In contrast
to the results from the competition experiment performed with
Gt and peptide 2 (Fig. 4c), the observed initial rates of MII
formation in this set of measurements were identical over the
Figure 6. MII stabilization by peptide 4, the F64Bpa substituted Gtc(60-71)far analog, measured by the ‘‘Extra MII’’ assay. ‘‘Extra MII’’
formation by increasing concentrations of peptide 4 (a). Dose–response curves of the ‘‘Extra MII’’ amplitudes of peptide 4 and for comparison
Gtc(60-71)far peptide (b). Shown are averages of three different measurements. Error bars indicate SEM.

Photochemistry and Photobiology, 2008, 84 837
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whole measured peptide concentration range. This indicates
that conformational changes in rhodopsin were rate-limiting
and that MII was formed from its precursor MI.
There are other examples in the literature where modifica-
tion with Bpa slightly increased ligand affinity for the
respective receptor. An N-formyl peptide ligand modified with
a Bpa moiety was reported to have a two-fold increased
affinity for its human neutrophil formyl peptide receptor (42).
Also, the attachment of a benzophenone probe to Ginkgolide
B at the 10-OH group, via either a methylene or carboxy
linkage, increased the ligand’s ability to inhibit the platelet-
activating factor receptor by more than three times (43).
CONCLUSION
We found that substituting Phe-64 of the Gtc(60-71)far peptide
by 4-benzoyl-L-phenylalanine (Bpa) yields a photolabile pep-
tide which retains its affinity for the active MII state. This
peptide should be suited to determine the binding site of
Gtc(60-71)far on the activated receptor by photocrosslinking
and subsequent receptor fragmentation followed by mass
spectrometric analysis. The great advantage of this novel MII-
recognizing peptide is the pentadeuterated benzophenone
moiety, which can be readily used in a mixture with the
nondeuterated twin peptide. This will lead to a facilitated MS
analysis of crosslinked samples, as labeled fragments of the
digested receptor will appear as double peaks in the MS
spectra. These characteristics make such peptides excellent
candidates for protein labeling studies in G protein research
and more general applications such as protein–protein inter-
action studies.
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Acknowledgements—We thank Klaus Peter Hofmann for helpful
discussions and critical reading of the manuscript. We acknowledge
support from NIH grants GM 36564 (to K.N.) and DFG Sfb449 (to
O.P.E.).
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